Stimulator Of Interferon Genes Agonists Research Articles

Sting Activation in Bone Marrow Macrophages Targets AML Blasts for Phagocytosis in an ICAM-1 Dependent Mechanism

The initiation and proliferation of acute myeloid leukaemia (AML) is in part regulated by the bone marrow microenvironment. Previous work by our group has shown that the stimulator of interferon genes (STING) pathway is activated in macrophages in the AML bone marrow microenvironment (Moore et al., 2022) . We found that AML-derived mitochondrial damage-associated molecular patterns were processed by bone marrow macrophages (BMMs) via LC3-associated phagocytosis (LAP). Furthermore, activation of STING resulted in a suppression of AML growth through an LAP dependent mechanism. Here we have investigated how the activation of STING in bone marrow macrophages increases macrophage phagocytic activity and in turn supresses the growth of AML. To understand the role of STING activation in bone marrow derived macrophages (BMDMs) in AML we used various STING agonists including DMXAA, 2'3'-cGAMP, and CpG oligonucleotides. We showed STING activation was induced through these agonists by measuring the upregulation of STING related genes ( Gbp2, Irf7 and Ifit3). We demonstrated that STING activation caused increased phagocytic clearance of live MN1 cells (mouse AML cell line) in an in vitro phagocytosis assay. In order to investigate how STING activation increases the phagocytic clearance of AML cells by BMDMs, we used three complementary methods. First, we performed a receptor tyrosine kinase array to measure the phosphorylation of mouse kinases following STING activation. Second, we investigated the transcriptomic changes resulting from STING activation with mRNA-sequencing. Third, after combining these results we identified an upregulation of key phagocytic pathways that specifically led us to focus on the adhesion molecules, in particular ICAM-1. Further investigation using an anti-ICAM-1 blocking antibody showed a reduction in the phagocytic potential of the BMDMs demonstrating the role of ICAM-1 in the phagocytic clearance of AML. Next, we activated human macrophages (derived from peripheral blood monocytes collected from healthy donors with approval of the East of England - Cambridgeshire and Hertfordshire Research Ethics Committee and the UK Health Research Authority, IRAS Project ID 33753) through STING which depleted AML in vitro confirming the results obtained using the mouse system. To investigate the in vivo significance of these findings, we treated C57Bl/6 animals with DMXAA and observed a significantly decreased tumour burden and increased survival following engraftment with MN1 cells. Cytotoxic T cell activity and number in the bone marrow was unchanged by STING activation in this model. In summary, we have shown that the activation of STING in bone marrow macrophages directly supresses the growth and proliferation of AML by stimulating the phagocytic activity of the macrophages, and the clearance of the leukaemic cells.

Bioactivable STING Nanoagonists to Synergize NIR-II Mild Photothermal Therapy Primed Robust and Long-Term Anticancer Immunity.

Pharmacological activation of the stimulator of interferon genes (STING) pathway has become a promising strategy for cancer immunotherapy. However, the insufficient tumorous accumulation, rapid clearance, and short duration of drug efficacy in the tumor microenvironment of small structural STING agonists greatly compromise the therapeutic efficacy. Herein, a tumorous extracellular matrix (ECM) is presented anchoring STING agonist-based photoimmunothernostic nanomedicine (SAPTN) that can be activated by mild-temperature photothermal therapy (mild PTT) induced neutrophilic inflammation. The SAPTN owns second window near-infrared (NIR-II) photonics properties fitting for NIR-II fluorescence and photoacoustic imaging-guided cancer therapy. The aggregates SAPTN targeting to the ECM provide slow and continuous release of potent STING agonists diABZIs. The mild PTT and long-lasting STING agonists released in the ECM synergisticallyprime systematic, robust, and long-term anticancer immunity. In a tumor model, this approach leads to complete tumor eradication in about 100% of mice with orthotopic breast tumors, and the mice regained tumor-free survival of at least 2 months. In addition, the immune-mediated abscopal effect shows inhibition of the distant solid tumor growth by intratumoral administration of SAPTN with laser irradiation. Overall, this approach represents a generalized photoactivable nanomedicine to prime anticancer immunity for improved cancer theranostics.

Characterizing the Distribution of a Stimulator of Interferon Genes Agonist and Its Metabolites in Mouse Liver by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry.

A STING (stimulator of interferon genes) agonist GSK3996915 under investigation in early discovery for hepatitis B was orally dosed to a mouse model for understanding the parent drug distribution in liver, the target organ. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to quantify the distribution of GSK3996915 in liver collected from mice administered a single oral dose at 90 mg/kg. GSK3996915 was detected with a zonal distribution localized in the portal triad and highly concentrated in the main bile ducts, indicating clearance through biliary excretion. High spatial resolution imaging showed the distribution of the parent drug localized to the cellular populations in the sinusoids, including the Kupffer cells. Additionally, a series of drug-related metabolites were observed to be localized in the central zones of the liver. These results exemplify the potential of utilizing MALDI IMS for measuring not only quantitative drug distribution and target exposure but also drug metabolism and elimination in a single suite of experiments. SIGNIFICANCE STATEMENT: An integrated imaging approach utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) complemented with immunohistochemistry (IHC) and histology was used to address the question of target exposure at the cellular level. Localized quantification of the parent drug in the target organ and identification of potential metabolites in the context of tissue histology were also achieved in one experimental suite to support characterization of pharmacokinetic properties of the drug in the early discovery stage.

Injectable Zwitterionic Physical Hydrogel with Enhanced Chemodynamic Therapy and Tumor Microenvironment Remodeling Properties for Synergistic Anticancer Therapy.

Surgical resection is the first-line therapy for breast cancer. However, residual tumor cells and the highly immunosuppressive tumor microenvironment (TME) continue to have a serious impact on tumor recurrence and metastasis postresection. Implantation of an in situ hydrogel system postresection has shown to be an effective treatment with great clinical potential. Herein, an injectable zwitterionic hydrogel system was developed for local drug delivery with enhanced immune activation and prevention of tumor recurrence. Driven by electrostatic interactions, poly(sulfobetaine methacrylate) (PSBMA) self-assembles into a hydrogel in saline, achieving low protein adsorption and tunable biodegradability. The chemotherapy drug doxorubicin (DOX) was loaded into copper peroxide nanoparticles (CuO2/DOX), which were coated with macrophage membranes to form tumor-targeting nanoparticles (M/CuO2/DOX). Next, M/CuO2/DOX and the stimulator of interferon genes (STING) agonist 2',3'-cGAMP were coloaded into PSBMA hydrogel (Gel@M/CuO2/DOX/STING). The hydrophilic STING agonist was first released by diffusion from hydrogel to activate the STING pathway and upregulate interferon (IFN) signaling related genes, remodeling the immunosuppressive TME. Then, M/CuO2/DOX targeted the residual tumor cells, combining with DOX-induced DNA damage, immunogenic tumor cell death, and copper death. Hence, this work combines chemodynamic therapy with STING pathway activation in TME, encouraging residual tumor cell death, promoting the maturation of dendritic cells, enhancing tumor-specific CD8+ T cell infiltration, and preventing postoperative recurrence and metastasis.

Mannose-functionalization of reconstituted high-density lipoprotein nanoparticles improves payload delivery and enhances M2-to-M1 phenotype reprogramming of RAW 264.7 macrophages polarized by B16-F10 melanoma cells

The targeting and conversion of the immunosuppressive (M2) tumor-associated macrophages (TAMs) to an immunostimulatory (M1) phenotype can induce tumor regression in advanced melanoma. We have previously characterized and reported the ability of reconstituted high-density lipoprotein nanoparticles (rHDL NPs) functionalized with DSPE-PEG-mannose (DPM) to deliver payload to macrophages. Herein, we investigate the modulation of macrophage phenotype and payload delivery mechanisms of the rHDL-DPM NPs in RAW 264.7 murine macrophages exposed to the conditioned medium (CM) from murine B16-F10 melanoma cells. The rHDL-DPM NPs loaded with the Stimulator of Interferon genes agonist, DMXAA, reduced protein levels of M2 markers. Through the mannose moiety, the rHDL-DPM-DMXAA NPs enhanced the production of interferon β and CXCL10 compared to the free DMXAA in the B16-F10 CM-educated RAW 264.7 macrophages. Compared to their non-mannosylated counterpart, the rHDL-DPM NPs delivered their payload more efficiently to the B16-F10 CM-educated RAW 264.7 macrophages. Mechanistically, both the scavenger receptor type B class 1 (SR-B1) and the mannose receptor (CD206) facilitated payload delivery to the macrophages via endocytic and non-endocytic mechanisms. Finally, the CM from rHDL-DPM-DMXAA NPs -treated macrophages enhanced paclitaxel (paclitaxel)-mediated cytotoxicity in B16-F10 cells. Together, these in vitro findings demonstrate the potential of the mannose-functionalized rHDL NPs in improving the targeting of M2-like TAMs and treatment outcomes when combined with immunotherapy or PTX in B16-F10 melanoma in vivo models.

A Non-Nucleotide STING Agonist MSA-2 Synergized with Manganese in Enhancing STING Activation to Elicit Potent Anti-RNA Virus Activity in the Cells.

Both Manganese (Mn2+) and MSA-2 can activate the downstream signal pathway through stimulator of interferon genes (STING) and induce the expression of type I interferon, which is important for hosts to protect against DNA viruses. However, its effect on RNA viruses remains unknown. In this study, we used Seneca Valley virus (SVV) as a model RNA virus to investigate the inhibitory effects of Mn2+ and MSA-2 on the virus replication in the porcine cells (PK-15 cells). The results showed that both MSA-2 and Mn2+ were able to inhibit the SVV replication in PK-15 cells. The combination of MAS-2 and Mn2+ could confer better protection against SVV. Further studies showed that MSA-2 and Mn2+ could activate TBK1, IRF3 and NFκB through STING and induce the expression of IFN-β, IL-6 and TNF-α. The present study confirmed that MSA-2 synergized with Mn2+ in STING activation to generate a better antiviral effect in vitro, which would be helpful for the further development of effective antiviral drugs in the future.

Chemical Biology Perspectives on STING Agonists as Tumor Immunotherapy.

Stimulator of interferon genes (STING) is a crucial adaptor protein in the innate immune response. STING activation triggers cytokine secretion, including type I interferon and initiates T cell-mediated adaptive immunity. The activated immune system converts "cold tumors" into "hot tumors" that are highly responsive to T cells by recruiting them to the tumor microenvironment, ultimately leading to potent and long-lasting antitumor effects. Unlike most immune checkpoint inhibitors, STING agonists represent a groundbreaking class of innate immune agonists that hold great potential for effectively targeting various cancer populations and are poised to become a blockbuster in tumor immunotherapy. This review will focus on the correlation between the STING signaling pathway and tumor immunity, as well as explore the impact of STING activation on other biological processes. Ultimately, we will summarize the development and optimization of STING agonists from a medicinal chemistry perspective, evaluate their potential in cancer therapy, and identify possible challenges for future advancement.

Loops Mediate Agonist-Induced Activation of the Stimulator of Interferon Genes Protein.

The stimulator of interferon genes (STING) is an important therapeutic target for cancer diseases. The activated STING recruits downstream tank-binding kinase 1 (TBK1) to trigger several important immune responses. However, the molecular mechanism of how agonist molecules mediate the STING-TBK1 interactions remains elusive. Here, we performed molecular dynamics simulations to capture the conformational changes of STING and TBK1 upon agonist binding. Our simulations revealed that multiple helices (α5-α7) and especially three loops (loop 6, loop 8, and C-terminal tail) of STING participated in the allosteric mediation of the STING-TBK1 interactions. Consistent results were also observed in the simulations of the constitutive activating mutant of STING (R284S). We further identified α5 as a key region in this agonist-induced activation mechanism of STING. Free-energy perturbation calculations of multiple STING agonists demonstrated that an alkynyl group targeting α5 is a determinant for agonist activities. These results not only offer deeper insights into the agonist-induced allosteric mediation of STING-TKB1 interactions but also provide a guidance for future drug development of this important therapeutic target.

Discovery of novel amidobenzimidazole derivatives as orally available small molecule modulators of stimulator of interferon genes for cancer immunotherapy

Stimulator of interferon genes (STING) agonists show promise as immunomodulatory agents for cancer therapy. In this study, we report the discovery of a novel orally available STING agonist, SAP-04, that exhibits potent immunomodulatory effects for cancer therapy. By optimizing the amidobenzimidazole core with various pyridine-based heterocyclic substituents, we identified a monomeric variant that displayed more efficient STING agonistic activity than the corresponding dimer. SAP-04 efficiently induced cytokine secretion related to innate immunity by directly binding of the compound to the STING protein, followed by sequential signal transduction for the STING signaling pathway and type I interferon (IFN) responses. Further pharmacological validation in vitro and in vivo demonstrated the potential utility of SAP-04 as an immunomodulatory agent for cancer therapy in vivo. The in vivo anticancer effect was observed in a 4T1 breast tumor syngeneic mouse model through oral administration of the compound. Our findings suggest a possible strategy for developing synthetically accessible monomeric variants as orally available STING agonists.

Synergistic immunostimulation for tumor sensitization with a biomineralized DNA sponge

The activation of stimulator of interferon genes (STING) plays a key role for provoking effective innate and adaptive immunity, yet is constrained by the limited clinical translation of STING agonists. Herein, we construct a smart DNA sponge to deliver STING agonists for robust cancer immunotherapy via the synergistic activation of innate immune pathways. The compact DNA sponge (Mn-CpG), assembling through a simple rolling circle amplification reaction, is encoded with numerous STING agonist (Mn2+) and toll-like receptor 9 (TLR9) agonist (CpG oligodeoxynucleotide) and allows their on-demand cellular delivery We show that Mn-CpG sponge can trigger the synergistic type I interferon response and robust T cell priming through a convenient coordination of different signaling pathways. In a poorly immunogenic breast cancer model, the method achieves the boosted immune responses with significantly inhibited tumor growth and metastasis.

Remodeling of Tumor Microenvironment by Nanozyme Combined cGAS-STING Signaling Pathway Agonist for Enhancing Cancer Immunotherapy.

Nanozymes and cyclic GMP-AMP synthase (cGAS) the stimulator of interferon genes (STING) signaling pathway, as powerful organons, can remodel the tumor microenvironment (TME) to increase efficacy and overcome drug resistance in cancer immunotherapy. Nanozymes have the potential to manipulate the TME by producing reactive oxygen species (ROS), which lead to positive oxidative stress in tumor cells. Cyclic dinucleotide (2',3'-cGAMP), as a second messenger, exists in the TME and can regulate it to achieve antitumor activity. In this work, Co,N-doped carbon dots (CoNCDs) were used as a model nanozyme to evaluate the properties of the anti-tumor mechanism, and effective inhibition of S180 tumor was achieved. Based on CoNCDs' good biocompatibility and therapeutic effect on the tumor, we then introduced the cGAS-STING agonist, and the combination of the CoNCDs and STING agonist significantly inhibited tumor growth, and no significant systemic toxicity was observed. The combined system achieved the enhanced tumor synergistic immunotherapy through TME reprogramming via the peroxidase-like activity of the CoNCDs and cGAS-STING signaling pathway agonist synergistically. Our work provides not only a new effective way to reprogram TME in vivo, but also a promising synergic antitumor therapy strategy.

Sonodynamic amplification of cGAS-STING activation by cobalt-based nanoagonist against bone and metastatic tumor

The therapeutic effect of cancer immunotherapy is restrained by limited patient response rate caused by ‘cold’ tumors with an intrinsically immunosuppressive tumor microenvironment (TME). Activating stimulator of interferon genes (STING) confers promising antitumor immunity even in ‘cold’ tumors, but the further promotion of STING agonists is hindered by undesirable toxicity, low specificity and lack of controllability. Herein, an ultrasound-controllable cGAS-STING amplifying nanoagonist was constructed by coordinating mitochondria-targeting ligand triphenylphosphonium (TPP) to sonodynamic cobalt organic framework nanosheets (TPP@CoTCPP). The Co ions specifically amplify STING activation only when cytosolic mitochondrial DNA leakage is caused by sonocatalysis-induced ROS production and sensed by cGAS. A series of downstream innate immune proinflammatory responses induced by local cGAS-STING pathway activation under spatiotemporal ultrasound stimulation efficiently prime the antitumor T-cell response against bone metastatic tumor, a typical immunosuppressive tumor. We also found that the coordination of TPP augments the sonodynamic effect of CoTCPP nanosheets by reducing the band gap, improving O2 adsorption and enhancing electron transfer. Overall, our study demonstrates that the targeted and amplified cGAS-STING activation in cancer cell controlled by spatiotemporal ultrasound irradiation boosts high-efficiency sonodynamic-ionicimmunotherapy against immunosuppressive tumor.

Antitumor effect of anti-vascular therapy with STING agonist depends on the tumor microenvironment context.

Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models. Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1). The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed. The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results.

Monitoring Melanoma Responses to STING Agonism and Focused Ultrasound Thermal Ablation Using Microneedles and Ultrasensitive Single Molecule Arrays

AbstractReal‐time monitoring of immune state and response to therapy can provide a means to stratify patients and increase the number of responders who will benefit from approved and emerging immunotherapies. The accessibility of immune cells in the skin provides an opportunity for local immune modulation as well as for noninvasive sampling of disease biomarkers in the skin interstitial fluid (ISF). Here, a monitoring strategy for melanoma immunotherapy is investigated by longitudinal sampling of biomarkers in the skin ISF using Hyaluronic acid (HA)‐based microneedles (MNs). Focused ultrasound ablation and delivery of nanoparticulate stimulator of interferon genes agonist are used as model immunotherapies. It is shown that this combination therapy induces potent inflammatory responses in a melanoma mouse model, promoting tumor elimination and immune memory formation. Indeed, quantifying soluble, protein‐based biomarkers following therapy using conventional immunoassay reveals a pronounced proinflammatory program in the tumor. However, conventional assays fail to detect the low concentration of biomarkers in plasma and in MN‐sampled ISF. It is shown that ultrasensitive single molecule arrays (Simoa) effectively detected proinflammatory biomarkers that are upregulated in response to the therapy in MN‐sampled ISF, in plasma, and in tumors, supporting the feasibility of monitoring melanoma immunotherapy using MNs.

Human STING is a proton channel.

Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.

Arabinose- and xylose-modified analogs of 2′,3′-cGAMP act as STING agonists

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containingarabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-β secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Reovirus combined with a STING agonist enhances anti-tumor immunity in a mouse model of colorectal cancer.

Reovirus, a naturally occurring oncolytic virus, initiates the lysis of tumor cells while simultaneously releasing tumor antigens or proapoptotic cytokines in the tumor microenvironment to augment anticancer immunity. However, reovirus has developed a strategy to evade antiviral immunity via its inhibitory effect on interferon production, which negatively affects the induction of antitumor immune responses. The mammalian adaptor protein Stimulator of Interferon Genes (STING) was identified as a key regulator that orchestrates immune responses by sensing cytosolic DNA derived from pathogens or tumors, resulting in the production of type I interferon. Recent studies reported the role of STING in innate immune responses to RNA viruses leading to the restriction of RNA virus replication. In the current study, we found that reovirus had a reciprocal reaction with a STING agonist regarding type I interferon responses in vitro; however, we found that the combination of reovirus and STING agonist enhanced anti-tumor immunity by enhancing cytotoxic T cell trafficking into tumors, leading to significant tumor regression and survival benefit in a syngeneic colorectal cancer model. Our data indicate the combination of reovirus and a STING agonist to enhance inflammation in the tumor microenvironment might be a strategy to improve oncolytic reovirus immunotherapy.

T-cell derived extracellular vesicles prime macrophages for improved STING based cancer immunotherapy.

A key phenomenon in cancer is the establishment of a highly immunosuppressive tumour microenvironment (TME). Despite advances in immunotherapy, where the purpose is to induce tumour recognition and hence hereof tumour eradication, the majority of patients applicable for such treatment still fail to respond. It has been suggested that high immunological activity in the tumour is essential for achieving effective response to immunotherapy, which therefore have led to exploration of strategies that triggers inflammatory pathways. Here activation of the stimulator of interferon genes (STING) signalling pathway has been considered an attractive target, as it is a potent trigger of pro-inflammatory cytokines and types I and III interferons. However, immunotherapy combined with targeted STING agonists has not yielded sustained clinical remission in humans. This suggests a need for exploring novel adjuvants to improve the innate immunological efficacy. Here, we demonstrate that extracellular vesicles (EVs), derived from activated CD4+ T cells (T-EVs), sensitizes macrophages to elevate STING activation, mediated by IFNγ carried on the T-EVs. Our work support that T-EVs can disrupt the immune suppressive environment in the tumour by reprogramming macrophages to a pro-inflammatory phenotype, and priming them for a robust immune response towards STING activation.

Discovery of BI 7446: A Potent Cyclic Dinucleotide STING Agonist with Broad-Spectrum Variant Activity for the Treatment of Cancer.

Activating the stimulator of interferon genes (STING) pathway with STING agonists is an attractive immune oncology concept to treat patients with tumors that are refractory to single-agent anti-PD-1 therapy. For best clinical translatability and broad application to cancer patients, STING agonists with potent cellular activation of all STING variants are desired. Novel cyclic dinucleotide (CDN)-based selective STING agonists were designed and synthesized comprising noncanonical nucleobase, ribose, and phosphorothioate moieties. This strategy led to the discovery of 2',3'-CDN 13 (BI 7446), which features unprecedented potency and activates all five STING variants in cellular assays. ADME profiling revealed that CDN 13 has attractive drug-like properties for development as an intratumoral agent. Injection of low doses of CDN 13 into tumors in mice induced long-lasting, tumor-specific immune-mediated tumor rejection. Based on its compelling preclinical profile, BI 7446 has been advanced to clinical trials (monotherapy and in combination with anti-PD-1 antibody).

Cyto-IL-15 synergizes with the STING agonist ADU-S100 to eliminate prostate tumors and confer durable immunity in mouse models.

Prostate cancer is one of the most commonly diagnosed malignancies in men with high mortality rates. Despite the recent therapeutic advances, such as immunotherapies, survival of patients with advance disease remains significantly low. Blockade of immune checkpoints has led to low response rates in these patients probably due to the immunosuppressive microenvironment and low mutation burden of prostate tumors. Combination of multiple immunotherapeutic regimes has also been unsatisfactory due to augmented adverse effects. To activate multiple immune-stimulatory pathways in the hostile prostate cancer microenvironment, we used a combination of cytotopically modified interleukin-15 (cyto-IL-15) with the stimulator of interferon genes (STING) agonist, ADU-S100. To determine whether this combination regime could lead to both local and systemic anti-tumor effects, intratumoral administration of these agents was used in murine models of prostate cancer. Tumor growth and mouse survival were monitored, and ex vivo analyses, and RNA sequencing were performed on the tumors. Intratumorally injected ADU-S100 and cyto-IL-15 synergized to eliminate tumors in 58-67% of mice with unilateral tumors and promoted abscopal immunity in 50% of mice with bilateral tumors treated only at one side. Moreover, this combination regime offered immunoprotection against tumor rechallenge in 83% of cured mice. The efficacy of the combination treatment was associated with a strong innate and adaptive immune activation and induction of apoptotic and necrotic cell death. Cytokines, including type I and II interferons, and cytokine signalling pathways were activated, NK and T cell mediated cytotoxicity was increased, and B cells were activated both locally and systemically. While ADU-S100 led to an ulcerative pathology at the injection site, no other adverse effects were observed. Localised administration of a STING agonist together with cyto-IL-15 can confer significant systemic benefits and long-lasting immunity against prostate tumors while reducing immune related toxicities.